Work is continuing on the purification to homogeneity of a unique inhibitor of adenylate cyclase (ACI). N.M.R., I.R., G.C.-Mass Spectroscopy, and possibly elemental analysis are the techniques that will be used to obtain information on the detailed chemical structure of this substance. Kinetic studies are being continued to elucidate the mechanism of action of ACI on adenylate cyclase. The inhibitor appears to act at the catalytic site of the enzyme. The nature of binding, the reversability of inhibition, and various other characteristics of the action of ACI are being investigated. The role of this inhibitor in various physiologic processes related to cyclic AMP mediated hormone responsiveness is being investigated. To measure the level of ACI under various conditions a radioimmunoassay will be developed. Utilizing infusion studies in the rat, additional studies will be done to determine the effects of the inhibitor in vivo on such cAMP triggered processes as glucagon-induced glycogenolysis, glucagon and catecholamine induced lipolysis, and hormone-mediated changes in serum and urinary cAMP levels. The levels of cyclic AMP and the inhibitor will be measured in the perfused rat liver preparations in response to hormones and exogenously administered dibutyryl cyclic AMP in normocalcemic and hypocalcemic conditions. The role of ACI in hormone refractioness will be investigated systematically.